In February 2012, FDA released its long-awaited draft guidance documents on biosimilars (1–3). The three documents outline the agency's thinking on the processes and requirements necessary to obtain approval for a biologic that could be demonstrated to be highly similar to an already marketed product. Central to the approval pathway is the idea that rigorous bioanalytical characterization of a proposed biosimilar would be the first step in showing that it is highly similar to a reference product. According to the draft guidance, the more comprehensive and robust the comparative structural and functional characterization of the innovator and proposed biosimilar, the more useful such characterization would be in determining what additional nonclinical and clinical studies might be needed for approval (1).

Looking for fingerprints

A key feature of the FDA draft guidance is the idea of a fingerprint-like identification of the protein, a set of quality attributes measured by orthogonal methods that in combination could be used to identify a protein and demonstrate similarity between a biosimilar and a comparator. This concept arose, in part, from experience in the approval of enoxaparin, a generic low-molecular weight heparin approved in July 2010. Enoxaparin was approved without a requirement for clinical trials, based on five criteria that the agency deemed sufficient to demonstrate that enoxaparin had the same active ingredient as the innovator product, Lovenox (4). These criteria were:

the physical and chemical characteristics of enoxaparin

the nature of the heparin material and the chemical process used to break up heparin chains into smaller pieces

Although enoxaparin is not a protein, it is a complex biologic product that required a level of physicochemical characterization similar to that for recombinant protein products.

Protein quality attributes: many and varied

Language in the draft guidance suggests that the level of information that could constitute a fingerprint would go beyond the basic information required of all protein products (see sidebar, "Protein Quality Attributes"). FDA states, "It may be useful to compare differences in the quality attributes of the proposed protein product with those of the reference product using a meaningful fingerprint-like analysis algorithm that covers a large number of additional product attributes and their combinations with high sensitivity using orthogonal methods (3)." Exactly what attributes of the protein the agency would like to see characterized, and what methods should be used are not defined, in part because of the great diversity in the properties of protein products.

"The fingerprint will depend on the individual molecule and the relevant critical quality attributes," says Fiona M. Greer, global director of BioPharma Services Development at SGS M-Scan. "It may be possible to standardize this for a particular class of molecule but probably not feasible between types of product. For example, glycosylation may have an effect on the efficacy in one type of molecule, but in another, changes in carbohydrate are not significant. Whatever the standard attributes are, they should cover both the physicochemical (primary and higher order structure) and biological properties. Without a doubt, the inclusion of information regarding posttranslational modifications will be required."

The fingerprint analogy can only be taken so far. Although real fingerprints are unique and unchanging, the attributes of a protein are harder to pin down. Protein products demonstrate variation in posttranslational modifications that may or may not be important for the biological activity of the protein. This type of heterogeneity is an expected feature of proteins produced in cultured cells, and the approval process for any biologic product typically includes a discussion with the regulatory agency where the range of heterogeneity is defined and limits agreed upon within which the potency and safety of the product remain unaffected.

For biosimilars manufacturers, determining the extent of variability in the comparator protein is an essential part of the characterization process. Typically, this step involves obtaining multiple batches of the comparator product manufactured at different times and evaluated at different points in their viable shelf life, and determining the range of quality attributes that the product displays.

"I wouldn't recommend picking a single batch and trying to produce a protein that's 99% identical to that batch," says Jin Xu, director of protein sciences at the Massachusetts Biomanufacturing Center. The comparator will exhibit a range of physicochemical properties that are acceptable, and the biosimilar has to fall within that range, explains Xu.